TWI410248B - Extract of leaves of typhonium blumei nicolson & sivadasan and its use - Google Patents

Abstract

The present invention is related to an extract of leaves of Typhonium blumei Nicolson & Sivadasan obtained by a process comprising steps of: first extraction, second extraction and third extraction, in which various extracted reagents including methanol, hexane and ethyl acetate are used respectively for extracting the leaves of Typhonium blumei Nicolson & Sivadasan. The extract of leaves of Typhonium blumei Nicolson & Sivadasan is capable of being used in manufacturing into medical assistants or functional products for improving hypertension and cardiovascular diseases.

Description

Soil summer leaf extract and its use

The present invention relates to a plant extract and its use, and in particular to a soil summer leaf extract and its use for improving hypertension or cardiovascular diseases and the like.

Hypertension is the primary threat to modern human health. Over the past few years, it has been ranked among the top ten causes of death in the country, and it has been linked to important adult diseases such as stroke, myocardial infarction and diabetes. The early symptoms of hypertension are not significant, and are often ignored and delayed to seek medical treatment, so that damage to major organs (such as the heart and aorta) is a serious threat to human health. Therefore, in order to improve the problem of hypertension in modern people, the administration and development of cardiovascular blood vessels are particularly important.

The clinical drug for the treatment of hypertension includes angiotensin-converting enzyme inhibitor (ACEi), such as Captopril, Enalapril, etc., and its pharmacological action is to inhibit vasoconstriction. Angiotensin-converting enZyme (ACE) activity reduces the production of angiotensin II and the hydrolysis of bradykinin, which causes vasodilation and blood volume to decrease, thereby achieving a blood pressure drop.

The development of ACEi-active foods has been an emerging trend in recent years. There have been reports of hydrolysates of food proteins such as zein (Miyoshi et al., 1991) and fish protein (Matsui et al., 1993; Nakajima et Al., 2009), soy protein (Wu et al., 2002) and noodle fruit (Yamaguchi et al., 2002), etc., all of which are functional foods that inhibit ACE activity; in addition, in Chinese herbal medicine, weeds (Rabdosia coetsa), Bambusae caulis, Ecklonia cava, Ligustrum vulgare L., Agapanthus africanus, Agave americana, yellow fennel (Clausena anisata), Dietes iridioides, and Tulbaghia violacea all have the effect of ACEi (Li et al., 2008, Phytomedicine 15: 386-388; Je et al., 2009, Food Chem. 113: 932-935; Wijesinghe et al., 2011, Nutrition Research and Practice 5(2): 93-100; Kiss et al., 2008. Journal of Ethnopharmacology 120(2): 220-225).

However, in the above-mentioned various ACEI active foods or Chinese herbal medicines, although the active ingredient which inhibits ACE, the inhibitory effect is often poor, or it cannot be separated and extracted efficiently, and the application level is limited.

Therefore, it is necessary to further develop natural herbaceous plants with ACEi activity, which are applied to the development of blood pressure lowering drugs, so as to effectively improve the problem of improving hypertension in Chinese people.

Typhonium blumei Nicolson & Sivadasan is a herbaceous plant of Araceae and Typhonium. Pinellia ternata is a traditional folklore herb, and its conventional use is used as an antidote or as a dressing for bruises and swelling, with dilatation, hemostasis, swelling, and detoxification. The leaves of Pinellia ternata are spicy and warm, and they are used to treat various cancers, cirrhosis and unknown swollen poisons. However, there is no report or application in inhibiting ACE activity.

The main object of the present invention is to provide a soil semi-summer leaf extract which has good angiotensin-converting enzyme inhibitor activity and is convenient for extraction and separation and can be applied to the development of a blood pressure lowering preparation or related health food.

The second object of the present invention is to provide the use of the extract of Pinellia ternata in improving blood pressure, cardiovascular diseases and the like, and the above-mentioned soil extract of Pinellia ternata as an active ingredient, which is applied to a blood pressure lowering auxiliary therapeutic agent or related health food. Development.

In order to achieve the foregoing object, the technical content of the present invention comprises: a soil summer leaf extract obtained by a method comprising the following steps: a first extraction step using ethanol as an extraction solvent, The soil summer leaves are subjected to extraction treatment so that the weight-to-volume ratio (w/v, g/ml) of the soil to the ethanol is 1:4 to 1:10, and the ethanol extract is recovered, and the ethanol extract is concentrated. Obtaining a first extract; and a second extracting step, using an equal volume of ethanol and n-hexane as an extraction solvent, extracting the first extract obtained in the first extraction step, and separating the layer to obtain an ethanol layer And concentrating under reduced pressure and lyophilizing to obtain a second extract, wherein the weight ratio of the first extract to the second extraction step extraction solvent is 1:1 to 1:10; and a third extraction step The second extract is added with an equal volume of water and ethyl acetate as an extraction solvent, and the second extract is subjected to extraction treatment, and the layer is separated, and the ethyl acetate layer is taken, and concentrated under reduced pressure and freeze-dried. Obtaining the soil Wherein the second extract with a volume ratio of extraction solvent weight ratio of 1: 1 to 1:10.

The soil summer leaf extract of the present invention, wherein the method further comprises a column chromatography step, after the third extraction step, the third extraction step of the soil, the summer leaf extract, using a rubber tube column Then, using n-hexane and ethyl acetate, and ethyl acetate and methanol as mobile phases, chromatographic separation treatment, collecting the washing liquid in a separate bottle, and analyzing the washing liquid by a film color layer, and combining the washing liquids having the same composition. Several fractions were obtained, which were numbered as Fr.1 to Fr.10 according to the washing sequence, and the Fr.10 was concentrated under reduced pressure and freeze-dried to obtain the soil summer leaf extract.

The soil summer leaf extract of the present invention, wherein the column chromatography step is performed by selecting the Fr. 2 to concentrate under reduced pressure and freeze-drying, thereby obtaining the soil summer leaf extract.

The soil summer leaf extract of the present invention, wherein the column chromatography step utilizes a mixture of different ratios of n-hexane and ethyl acetate, wherein the ratio of the n-hexane to the ethyl acetate is 100% n-hexane. Gradually change to 1:9 in a ratio of 10% each time, followed by a mixture of ethyl acetate and methanol in different proportions as a mobile phase, wherein the mixing ratio of ethyl acetate and methanol is 100% ethyl acetate, in order The ratio was gradually changed to 100% methanol at a rate of 10% each time for chromatographic separation.

The soil summer leaf extract of the present invention, wherein the mobile phase of each ratio is sequentially introduced into the rubber tube column in a volume of 500 ml, and the washing liquid is collected in a bottle under the rubber tube column, and the washing liquid is collected in each bottle. The amount of liquid is 250 ml.

The soil summer leaf extract of the present invention, wherein the extraction solvent of the first extraction step is 50% to 95% ethanol.

The soil summer leaf extract of the present invention, wherein the first extraction step, the second extraction step and the third extraction step are operated at an agitation rate of 100 to 1000 rpm, 25 ° C to 70 ° C, and repeated extraction 1 to 3 times, each extraction for more than 30 minutes.

The soil summer leaf extract of the present invention, wherein the first extraction step is repeated 3 times and extracted for 24 hours each time.

The soil summer leaf extract of the present invention, wherein the second extraction step is repeated 3 times and extracted for 3 hours each time.

The soil summer leaf extract of the present invention, wherein the third extraction step is repeated 3 times and extracted for 3 hours each time.

Further, the use of the soil summer leaf extract of the present invention is the use of the soil summer leaf extract as an active substance for the preparation of an adjuvant therapeutic or health food for improving hypertension.

The above and other objects, features and advantages of the present invention will become more <RTIgt;

Referring to FIG. 1 , the soil summer leaf extract of the first embodiment of the present invention is obtained by a method comprising the following steps: a first extraction step S1, a second extraction step S2 and a first Three extraction steps S3.

The first extraction step S1 of the present invention uses ethanol as an extraction solvent to extract the soil of Pinellia ternata, so that the weight-to-volume ratio (w/v, g/ml) of the soil to the ethanol is 1:4. 1:10, and the ethanol extract was recovered, and the ethanol extract was concentrated to obtain a first extract. In more detail, the first extraction step S1 is to prepare fresh soil Pinellia ternata, and after lyophilization by liquid nitrogen, the mash is used, and the first extraction step S1 uses 50% to 95% ethanol as an extraction solvent. The extraction is carried out for 30 minutes or more at an agitation rate of 100 to 1000 rpm, 25 to 70 ° C, and repeated extraction 1 to 3 times, preferably for 24 hours and repeated extraction 3 times, to recover an ethanol extract. The ethanol extract is further subjected to removal of an extraction solvent using a vacuum condenser, and then dried using a freeze dryer to obtain the first extract.

The second extraction step S2 uses an equal volume of ethanol and n-hexane as an extraction solvent, and the first extract obtained in the first extraction step S1 is subjected to extraction treatment, and after standing and layering, an ethanol layer is taken and concentrated under reduced pressure. And lyophilizing to obtain a second extract, wherein the weight ratio of the first extract to the extraction solvent is 1:1 to 1:10. More specifically, the second extraction step S2 is carried out in the first extract by adding an equal volume of ethanol and n-hexane as an extraction solvent, and extracting at an agitation rate of 100 to 1000 rpm and 25 to 70 ° C. More than 30 minutes, and repeated extraction 1 to 3 times, preferably 3 hours of extraction and repeated extraction 3 times, after standing and layering, separately collect the upper n-hexane layer and the lower ethanol layer, the n-hexane layer The n-hexane extract can be obtained by concentration under reduced pressure and freeze-drying; the ethanol layer is concentrated under reduced pressure and freeze-dried to obtain the second extract.

The third extraction step S3, the second extract is taken, and an equal volume of water and ethyl acetate are added as an extraction solvent for extraction treatment, wherein the weight ratio of the second extract to the extraction solvent is 1:1 to 1:10; After standing to separate the layers, an ethyl acetate layer was taken, and the ethyl acetate layer was concentrated to obtain a soil extract of Pinellia ternata. More specifically, the third extraction step S3 collects the second extract obtained in the second extraction step S2, adding an equal volume of water and ethyl acetate as an extraction solvent, and stirring at a rate of 100 to 1000 rpm. , the extraction temperature is 25 ° C to 70 ° C environment for more than 30 minutes, and repeated extraction 1 to 3 times, preferably for 3 hours and repeated extraction 3 times, after standing layering, take the upper ethyl acetate layer The extractive solvent of the first embodiment was obtained by removing the extraction solvent by a reduced pressure concentrator and then lyophilizing.

The soil semi-summer leaf extract of the present invention is subjected to solvent extraction treatment by the aforementioned method to obtain an active ingredient having an angiotensin-converting enzyme inhibitor activity in the soil, and can be further applied to a blood pressure auxiliary therapeutic agent. Or the development of health food. Please refer to the first table for the yield of the two extract samples and the angiotensin-converting enzyme inhibitor activity analysis, wherein the n-hexane extract is collected in the second extraction step S2 and freeze-dried. The n-hexane extract obtained by the treatment; the soil pinellia leaf extract is the soil summer leaf extract of the first embodiment of the present invention.

Among them, 75 μl of soil Pinellia ternata extract and n-hexane extract were thoroughly mixed with 75 μl of angiotensin Converting Enzyme form rabbit lung (ACE; Sigma Chemicals). After standing at 37 ° C for 10 minutes in a water bath, 75 μl of (μl) HHL substrate solution (Hippuryl-His-Leu acetate salt, HHL; Sigma Chemicals) was added to react in a 37 ° C water bath for 30 minutes, and finally 1 N was added. The reaction was terminated with hydrochloric acid (HCl). The hippuric acid (HA) produced by the reaction of each sample was extracted with 750 μl of ethyl acetate as an extraction solvent, and then centrifuged at 3,600 rpm for 1 minute after shaking. Five minutes, 500 μl (μl) of the supernatant after centrifugation was taken. The supernatant was evaporated to dryness in a water bath at 80 ° C, and dissolved in 1 ml (ml) of deionized water. Finally, the absorbance was measured by a spectrophotometer at a wavelength of 228 nm, and the obtained result was further brought into Formula 1, and each sample was calculated for the sample. Inhibitory ability of angiotensin converting enzyme.

Formula 1: Inhibition ability (%) = [(Ac-As) / (Ac-Ab)] × 100%; where As represents the absorbance measured after the reaction of each of the above extract samples, and Ac represents the extraction by buffer solution instead of extraction The absorbance measured after the reaction of the sample of the substance, Ab represents the absorbance value measured by the same detection step after adding the hydrochloric acid to terminate the reaction before each sample of the extract. The present invention is based on the IC ₅₀ value as an activity index of an angiotensin-converting enzyme inhibitor, and the significance of the IC ₅₀ value is that the measured extract sample can inhibit the sample concentration of the angiotensin converting enzyme activity by 50%. First, the sample of the extract to be tested is diluted to a different concentration with a buffer solution or a solvent such as DMSO (dimethyl sulfoxide), and the inhibition ability of the angiotensin-converting enzyme is determined by the above method, and the statistical regression line is obtained. Based on this regression equation, the concentration of the extract sample required to inhibit 50% of the angiotensin-converting enzyme activity was determined.

Table 1: Yield and ACEI activity of extract samples

From the IC ₅₀ value shown in the results of the first table, it was found that the soil summer leaf extract of the first embodiment of the present invention did have an activity of inhibiting angiotensin converting enzyme.

Please refer to FIG. 2, which is the effect of different concentrations of soil Pinellia ternata extract (the first summer leaf extract of the first embodiment of the present invention) on angiotensin-converting enzyme activity, and its IC can be calculated from its regression line. _{The 50} value is 494.4 μg/ml.

Referring to FIG. 3, the soil summer leaf extract of the second embodiment of the present invention is obtained by a method comprising the following steps: a first extraction step S4, a second extraction step S5, and a first extraction step S5. The third extraction step S6 and a column chromatography step S7.

Wherein the first, second and third extraction steps S4, S5, S6 and the first, second and third extraction steps S1, S2, S3 for preparing the soil summer leaf extract of the first embodiment are not Different, so no longer repeat them.

The chromatography step S7 is carried out by using a tantalum rubber tube column, using a ruthenium rubber column, sequentially using different ratios of n-hexane and ethyl acetate, and different ratios of ethyl acetate and methanol as mobile phases for chromatographic separation treatment. The washing liquid is collected in a divided bottle, and the washing liquid is analyzed by a film color layer, and the washing liquids having the same composition are combined to obtain a plurality of parts, and the parts are concentrated under reduced pressure and freeze-dried to obtain the second embodiment of the present invention. Example soil Pinellia leaf extract. Wherein, the film color layer analysis is carried out by using a mixture of n-hexane and ethyl acetate in a ratio of 6:4 or 100% methanol as a mobile phase, and analyzing the components of each bottle of washing liquid, thereby merging the washing liquids containing the same components. And obtain the plurality of distinctions.

More specifically, the column chromatography step S7 is a silica gel column with a length of 73 cm (cm) and a tube diameter of 6 cm (cm), which is a silica gel; Ultra Pure Silica Gel. Particle size 230-400 mesh, 40-63 um pore size, Silicycle Chemical Division, Canada) as a stationary phase, using a mixture of different ratios of n-hexane and ethyl acetate as the mobile phase, wherein the mixture of n-hexane and the ethyl acetate The ratio is gradually changed from 100% n-hexane to 10% in the ratio of 1:9, followed by a mixture of ethyl acetate and methanol in different proportions, wherein the mixing ratio of ethyl acetate and methanol is 100. Ethyl acetate was gradually changed to 100% methanol in a ratio of 10% each for separation by chromatography. Wherein, the mobile phases of each ratio are sequentially introduced into the cartridge column in 500 ml (ml), and the flow rate is decreased by about 2.5 cm (min) per minute, and the bottle is collected and washed at the outlet below the cartridge column. The amount of washing liquid received per bottle was 250 ml (ml). After the film color layer analysis and combination, a total of 10 distinguishing substances were obtained, which were numbered as Fr.1 to Fr.10 according to the washing sequence. After the concentration was concentrated under reduced pressure and freeze-dried, it was the first invention. The soil of the second embodiment of the Pinellia ternata extract.

In this embodiment, the extracts of the plurality of different parts, numbered as Fr. 2, Fr. 10 or a combination thereof, and the soil of the summer leaves obtained by concentration under reduced pressure and freeze-dried are preferably taken. The soil summer leaf extract can preferably inhibit angiotensin converting enzyme activity.

Referring to Table 2, the IC ₅₀ value for inhibiting angiotensin-converting enzyme activity of each of the above-mentioned different substances is the best, and the inhibitory activity of the No. Fr. 10 is the best, and the result shows the distinction of the second embodiment. The Fr.10 and Fr.2 do have the activity of an angiotensin-converting enzyme inhibitor, and the fraction can be applied to the development and preparation of a blood pressure-lowering auxiliary therapeutic agent or related health food after being concentrated under reduced pressure and freeze-dried to improve The problem of high blood pressure in Chinese people. The angiotensin-converting enzyme inhibitor activity analysis of this example is the same as that of the first embodiment, and is carried out by the calculation of the formula 1. Please refer to Fig. 4, which is the effect of different concentrations of the Fr.10 sample on the angiotensin-converting enzyme activity, and its IC ₅₀ value can be calculated from its regression line to be 383.7 μg/ml.

Table 2: ACEi activity of each component

Referring to Fig. 5, for the gas chromatographic analysis of the distinguishing material Fr. 10, the present invention selects a Varian 450-GC and a Varian 240-MS system (Varian, USA) for gas chromatography mass spectrometry ( GC-Mass), VF-5ms column (30 m × 0.25 mm, 0.25 μm, FactorFour ^TM , USA) with helium as the carrier gas, set at a gas flow rate of 1.0 ml (ml) per minute, shunt The analysis was carried out under the conditions of a ratio of 10:1 and an injection port temperature of 250 °C. Wherein, the temperature change of the column is set at 50 ° C for 5 minutes, then the temperature is raised to 120 ° C at a rate of 5 ° C per minute and maintained at 120 ° C for 8 minutes, and then at a rate of 8 ° C per minute. The temperature was raised to 200 ° C and finally the temperature was raised to 300 ° C at a rate of 3 ° C per minute and maintained for 10 minutes. The mass spectrometry collection range was set to 50-650 amu and the electron pulse voltage was 10e. The results obtained can be further verified in the National Institute of Standards and Technology (NIST MS 2.0 database, USA) database.

Please refer to the table 3 for the identification of the analysis by gas chromatography mass spectrometry. It shows that the Fr.10 contains at least 13 compounds, of which Octadecanoic acid, Hexadecanoic acid and its esters are mostly The content accounts for about 60% of the total.

Table 3: Chemical composition analysis results of the classification Fr.10

Please refer to Fig. 6 for the pharmacokinetic analysis results presented by the reference Fr. 10, which shows that the Fr. 10 line is a competitive inhibition type.

According to the above, the soil of the summer leaves of the present invention has good angiotensin-converting enzyme inhibitory activity, and thus has the effects of lowering blood pressure and preventing cardiovascular diseases, and can be used as an auxiliary therapeutic agent for hypertension or a health food. The soil Pinellia ternata extract may be used alone in the development of related adjuvant therapeutics or health foods, or may be combined with a pharmaceutically acceptable carrier, excipient or other nutrient composition. Wherein, the soil summer leaf extract system can be prepared into any convenient edible form, such as a tablet, a capsule, a powder, a granule or a liquid agent, or the soil of the Pinellia ternata extract is combined with other foods or beverages for further processing. Make a more food-like form for consumption for oral administration.

The soil summer leaf extract of the invention has good angiotensin-converting enzyme inhibitor activity and can be prepared through a simple extraction step, and can be applied to the research and development of blood pressure-related auxiliary therapeutic agents or health foods to improve modernity. The problem of human hypertension is the efficacy of the present invention.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

S1. . . First extraction step

S2. . . Second extraction step

S3. . . Third extraction step

S4. . . First extraction step

S5. . . Second extraction step

S6. . . Third extraction step

S7. . . Column chromatography step

Fig. 1 is a view showing the extraction method of the soil summer leaf extract of the first embodiment of the present invention.

Fig. 2 is a graph showing the analysis of ACEI activity of the Pinellia ternata extract of the first embodiment of the present invention.

Fig. 3 is a view showing the extraction method of the soil summer leaf extract of the second embodiment of the present invention.

Fig. 4 is a graph showing the analysis of the ACEI activity of the distinguishing substance Fr. 10 of the second embodiment of the present invention.

Fig. 5 is a gas chromatographic mass spectrometry diagram of the distinguishing material Fr. 10 of the second embodiment of the present invention.

Fig. 6 is a diagram showing the enzyme kinetic inhibition type of the distinguishing substance Fr. 10 of the second embodiment of the present invention.

S1. . . First extraction step

S2. . . Second extraction step

S3. . . Third extraction step

Claims (11)

A soil Pinellia ternata extract is prepared by a method comprising the following steps: a first extraction step of extracting the soil of Pinellia ternata using ethanol as an extraction solvent to make the soil of the summer leaves and the ethanol The weight-to-volume ratio (w/v, g/ml) is 1:4 to 1:10, and the ethanol extract is recovered, and the ethanol extract is concentrated and lyophilized to obtain a first extract; a second extraction step, The first extract obtained in the first extraction step is subjected to extraction treatment by using an equal volume of ethanol and n-hexane as an extraction solvent, and the layer is separated after standing, and the ethanol layer is taken, concentrated under reduced pressure and freeze-dried to obtain a second. The extract, wherein the weight ratio of the first extract to the extraction solvent of the second extraction step is 1:1 to 1:10; and a third extraction step is to add an equal volume to the second extract. The second extract is subjected to extraction treatment by using water and ethyl acetate as an extraction solvent, and the layer is separated, and then the ethyl acetate layer is taken, and concentrated under reduced pressure and freeze-dried to obtain the soil extract of Pinellia ternata, wherein Second extract and the third extraction step Weight to volume ratio of extraction solvent is from 1: 1 to 1:10. The method of claim 19, wherein the method further comprises a column chromatography step, after the third extraction step, the soil of the third extraction step is extracted from the summer leaves. The product was subjected to chromatographic separation using a column of a rubber tube, followed by n-hexane and ethyl acetate, and ethyl acetate and methanol as a mobile phase, and the washing liquid was collected in a vial, and the washing liquid was analyzed by a film color layer. The washing liquids having the same composition are combined to obtain a plurality of distinguishing substances, which are numbered according to the washing order and are numbered as Fr.1 to Fr.10, and the Fr.10 is selected and concentrated under reduced pressure and freeze-dried. Soil pinellia leaf extract. The soil summer leaf extract according to claim 2, wherein the column chromatography step is performed after the Fr. 2 is concentrated under reduced pressure and freeze-dried to obtain the soil summer leaf extract. The soil summer leaf extract according to claim 2, wherein the column chromatography step uses a mixture of different ratios of n-hexane and ethyl acetate as a mobile phase, wherein the n-hexane and the acetic acid B The mixing ratio of the ester is gradually changed from 100% n-hexane to 10% in the ratio of 1:9, followed by a mixture of ethyl acetate and methanol in different proportions as a mobile phase, wherein the mixture of ethyl acetate and methanol is mixed. The ratio was gradually changed from 100% ethyl acetate to 10% methanol in the order of 10% each time for chromatographic separation. The soil summer leaf extract according to claim 4, wherein the liquid phase of each ratio is sequentially introduced into the rubber tube column in a volume of 500 ml, and the washing liquid is collected in a bottle under the rubber tube column. The amount of washing liquid collected per bottle is 250 ml. The soil summer leaf extract according to claim 1, wherein the extraction solvent of the first extraction step is 50% to 95% ethanol. The soil summer leaf extract according to claim 1, wherein the first extraction step, the second extraction step and the third extraction step are operated at a stirring rate of 100 to 1000 rpm, 25 to 70 ° C. Environment, repeated extraction 1 to 3 times, each extraction for more than 30 minutes. The soil summer leaf extract according to claim 7, wherein the first extraction step is repeated 3 times and extracted for 24 hours each time. The soil summer leaf extract according to claim 7, wherein the second extraction step is repeated 3 times and extracted for 3 hours each time. The soil summer leaf extract according to claim 7, wherein the third extraction step is repeated 3 times and extracted for 3 hours each time. The use of the soil summer leaf extract as described in claim 1 or 2, which is applied as an active substance to prepare an adjuvant therapeutic or health food for improving hypertension.